Riccardo Montioli

Molecular defects of the glycine 41 variants of alanine glyoxylate aminotransferase associated with primary hyperoxaluria type I

G41 is an interfacial residue located within the α-helix 34–42 of alanine:glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (∼5,000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 → R mutation causes a partial unwinding of the 34–42 α-helix and a displacement of the first 44 N-terminal residues including the active site loop 24–32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations.

Open conformation of human DOPA decarboxylase reveals the mechanism of PLP addition to Group II decarboxylases

DOPA decarboxylase, the dimeric enzyme responsible for the synthesis of neurotransmitters dopamine and serotonin, is involved in severe neurological diseases such as Parkinson disease, schizophrenia, and depression. Binding of the pyridoxal-5′-phosphate (PLP) cofactor to the apoenzyme is thought to represent a central mechanism for the regulation of its activity. We solved the structure of the human apoenzyme and found it exists in an unexpected open conformation: compared to the pig kidney holoenzyme, the dimer subunits move 20 Å apart and the two active sites become solvent exposed. Moreover, by tuning the PLP concentration in the crystals, we obtained two more structures with different conformations of the active site. Analysis of three-dimensional data coupled to a kinetic study allows to identify the structural determinants of the open/close conformational change occurring upon PLP binding and thereby propose a model for the preferential degradation of the apoenzymes of Group II decarboxylases.

Molecular Insight into the Synergism between the Minor Allele of Human Liver Peroxisomal Alanine:Glyoxylate Aminotransferase and the F152I Mutation

Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that converts glyoxylate into glycine. AGT deficiency causes primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, due to a marked increase in hepatic oxalate production. Normal human AGT exists as two polymorphic variants: the major (AGT-Ma) and the minor (AGT-Mi) allele. AGT-Mi causes the PH1 disease only when combined with some mutations. In this study, the molecular basis of the synergism between AGT-Mi and F152I mutation has been investigated through a detailed biochemical characterization of AGT-Mi and the Phe152 variants combined either with the major (F152I-Ma, F152A-Ma) or the minor allele (F152I-Mi). Although these species show spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma, the Phe152 variants exhibit the following differences with respect to AGT-Ma and AGT-Mi: (i) pyridoxamine 5′-phosphate (PMP) is released during the overall transamination leading to the conversion into apoenzymes, and (ii) the PMP binding affinity is at least 200–1400-fold lower. Thus, Phe152 is not an essential residue for transaminase activity, but plays a role in selectively stabilizing the AGT-PMP complex, by a proper orientation of Trp108, as suggested by bioinformatic analysis. These data, together with the finding that apoF152I-Mi is the only species that at physiological temperature undergoes a time-dependent inactivation and concomitant aggregation, shed light on the molecular defects resulting from the association of the F152I mutation with AGT-Mi, and allow to speculate on the responsiveness to pyridoxine therapy of PH1 patients carrying this mutation.

Identification by Virtual Screening and In Vitro Testing of Human DOPA Decarboxylase Inhibitors

Dopa decarboxylase (DDC), a pyridoxal 5′-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinsons disease (PD). PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtual-screening (VS) protocol by experimentally testing the “in vitro” activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with Ki values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with Ki values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a Ki value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

Biochemical analyses are instrumental in identifying the impact of mutations on holo and/or apo-forms and on the region(s) of alanine:glyoxylate aminotransferase variants associated with primary hyperoxaluria type I.

Primary Hyperoxaluria Type I (PH1) is a disorder of glyoxylate metabolism caused by mutations in the human AGXT gene encoding liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5′-phosphate (PLP) dependent enzyme. Previous investigations highlighted that, although PH1 is characterized by a significant variability in terms of enzymatic phenotype, the majority of the pathogenic variants are believed to share both structural and functional defects, as mainly revealed by data on AGT activity and expression level in crude cellular extracts. However, the knowledge of the defects of the AGT variants at a protein level is still poor. We therefore performed a side-by-side comparison between normal AGT and nine purified recombinant pathogenic variants in terms of catalytic activity, coenzyme binding mode and affinity, spectroscopic features, oligomerization, and thermal stability of both the holo- and apo-forms. Notably, we chose four variants in which the mutated residues are located in the large domain of AGT either within the active site and interacting with the coenzyme or in its proximity, and five variants in which the mutated residues are distant from the active site either in the large or in the small domain. Overall, this integrated analysis of enzymatic activity, spectroscopic and stability information is used to (i) reassess previous data obtained with crude cellular extracts, (ii) establish which form(s) (i.e. holoenzyme and/or apoenzyme) and region(s) (i.e. active site microenvironment, large and/or small domain) of the protein are affected by each mutation, and (iii) suggest the possible therapeutic approach for patients bearing the examined mutations.

Molecular insights into the pathogenicity of variants associated with the aromatic amino acid decarboxylase deficiency

Dopa decarboxylase (DDC or AADC) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-aromatic amino acids into the corresponding aromatic amines. AADC deficiency is an inborn error of neurotransmitters biosynthesis with an autosomal recessive inheritance. About 30 pathogenic mutations have been identified, but the enzymatic phenotypes causing AADC deficiency are unknown, and the therapeutic management is challenging. Here, we report biochemical and bioinformatic analyses of the human wild-type DDC and the pathogenic variants G102S, F309L, S147R and A275T whose mutations concern amino acid residues at or near the active site. We found that the mutations cause, even if to different extents, a decreased PLP binding affinity (in the range 1.4-170-fold), an altered state of the bound coenzyme and of its microenvironment, and a reduced catalytic efficiency (in the range 17-930-fold). Moreover, as compared to wild-type, the external aldimines formed by the variants with L-aromatic amino acids exhibit different spectroscopic features, do not protect against limited proteolysis, and lead to the formation, in addition to aromatic amines, of cyclic-substrate adducts. This suggests that these external Schiff bases are not properly oriented and anchored, i.e., in a conformation not completely productive for decarboxylation. The external aldimines that the variants form with D-Dopa also appear not to be correctly located at their active site, as suggested by the rate constants of PLP-L-Dopa adduct production higher than that of the wild-type. The possible therapeutic implications of the data are discussed in the light of the molecular defects of the pathogenic variants.

Liver peroxisomal alanine:glyoxylate aminotransferase and the effects of mutations associated with Primary Hyperoxaluria Type I: An overview.

Liver peroxisomal alanine:glyoxylate aminotransferase (AGT) (EC 2.6.1.44) catalyses the conversion of l-alanine and glyoxylate to pyruvate and glycine, a reaction that allows glyoxylate detoxification. Inherited mutations on the AGXT gene encoding AGT lead to Primary Hyperoxaluria Type I (PH1), a rare disorder characterized by the deposition of calcium oxalate crystals primarily in the urinary tract. Here we describe the results obtained on the biochemical features of AGT as well as on the molecular and cellular effects of polymorphic and pathogenic mutations. A complex scenario on the molecular pathogenesis of PH1 emerges in which the co-inheritance of polymorphic changes and the condition of homozygosis or compound heterozygosis are two important factors that determine the enzymatic phenotype of PH1 patients. All the reported data represent relevant steps toward the understanding of genotype/phenotype correlations, the prediction of the response of the patients to the available therapies, and the development of new therapeutic approaches. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

S81L and G170R mutations causing Primary Hyperoxaluria type I in homozygosis and heterozygosis: an example of positive interallelic complementation

Primary Hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal-5′-phosphate (PLP) enzyme present in humans as major (Ma) and minor (Mi) allele. PH1-causing mutations are mostly missense identified in both homozygous and compound heterozygous patients. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, whereas the molecular aspects of the genotype-enzymatic-clinical phenotype relationship in compound heterozygous patients are completely unknown. Here, for the first time, we elucidate the enzymatic phenotype linked to the S81L mutation on AGT-Ma, relative to a PLP-binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functionality, is present in the second allele. By using a bicistronic eukaryotic expression vector, we demonstrate that (i) S81L-Ma is mainly in its apo-form and has a significant peroxisomal localization and (ii) S81L and G170R monomers interact giving rise to the G170R-Mi/S81L-Ma holo-form, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the heterodimer and the homodimeric counterparts in their purified recombinant form, (i) highlight the molecular basis of the pathogenicity of S81L-Ma and (ii) provide evidence for a positive interallelic complementation between the S81L and G170R monomers. Our study represents a valid approach to investigate the molecular pathogenesis of PH1 in compound heterozygous patients.

The N-terminal extension is essential for the formation of the active dimeric structure of liver peroxisomal alanine:glyoxylate aminotransferase.

Alanine:glyoxylate aminotransferase (AGT) is a pyridoxal-phosphate (PLP)-dependent enzyme. Its deficiency causes the hereditary kidney stone disease primary hyperoxaluria type 1. AGT is a highly stable compact dimer and the first 21 residues of each subunit form an extension which wraps over the surface of the neighboring subunit. Naturally occurring and artificial amino acid replacements in this extension create changes in the functional properties of AGT in mammalian cells, including relocation of the enzyme from peroxisomes to mitochondria. In order to elucidate the structural and functional role of this N-terminal extension, we have analyzed the consequences of its removal using a variety of biochemical and cell biological methods. When expressed in Escherichia coli, the N-terminal deleted form of AGT showed the presence of the protein but in an insoluble form resulting in only a 10% soluble yield as compared to the full-length version. The purified soluble fraction showed reduced affinity for PLP and greatly reduced catalytic activity. Although maintaining a dimer form, it was highly prone to self-aggregation. When expressed in a mammalian cell line, the truncated construct was normally targeted to peroxisomes, where it formed large stable but catalytically inactive aggregates. These results suggest that the N-terminal extension plays an essential role in allowing AGT to attain its correct conformation and functional activity. The precise mechanism of this effect is still under investigation.

Biochemical and computational approaches to improve the clinical treatment of dopa decarboxylase-related diseases: an overview.

Dopa decarboxylase (DDC) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme that by catalyzing the decarboxylation of L-Dopa and L-5-hydroxytryptophan produces the neurotransmitters dopamine and serotonin. The functional properties of pig kidney and human DDC enzymes have been extensively characterized, and the crystal structure of the enzyme in the holo- and apo-forms has been elucidated. DDC is a clinically relevant enzyme since it is involved in Parkinson’s disease (PD) and in aromatic amino acid decarboxylase (AADC) deficiency. PD, a chronic progressive neurological disorder characterized by tremor, bradykinesia, rigidity and postural instability, results from the degeneration of dopamine-producing cells in the substantia nigra of the brain. On the other hand, AADC deficiency is a rare debilitating recessive genetic disorder due to mutations in AADC gene leading to the inability to synthesize dopamine and serotonin. Development delay, abnormal movements, oculogyric crises and vegetative symptoms characterize this severe neurometabolic disease. This article is an up to date review of the therapies currently used in the treatment of PD and AADC deficiency as well as of the recent findings that, on one hand provide precious guidelines for the drug development process necessary to PD therapy, and, on the other, suggest an aimed therapeutic approach based on the elucidation of the molecular defects of each variant associated with AADC deficiency.